The broad, long-term goal of our research program is to advance knowledge of virus-host cell interactions by characterizing cellular cofactors essential for viral infections, particularly positive-strand RNA viruses such as hepatitis C virus (HCV) and dengue virus (DENV). These viruses infect millions of people worldwide and pose major threats to human health. Understanding how these viruses interact with the host cell is of critical importance to the development of diagnostics, antiviral drugs, and a prophylactic vaccine. In preliminary studies, we have uncovered a critical role of lipid droplets and one of its associated proteins in DENV and HCV entry into host cells at the membrane fusion step. Although a role of the lipid droplets in viral assembly has been reported, a function of these organelles in viral entry and membrane fusion has not been proposed before. In this study, we will investigate the mechanism of action for lipid droplets and its associated protein, CIDEB, to facilitate the entry of diverse RNA viruses. The following specific aims are proposed: (1) to define the roles of lipid droplets and their recruitment of early endosomes in virus entry. Our working hypothesis of this aim is that LDs and the recruitment of virus-containing early endosomes to LDs are required for completing viral entry. We will use pharmacological and genetic manipulations to reduce LDs in the target cells of DENV and HCV and then determine if infection by these viruses is inhibited at specific steps such as endocytosis, membrane fusion, or capsid release. (2) to determine the function of CIDEB during the infection cycle of positive-strand RNA viruses. We have demonstrated the CIDEB knockdown (KD) with RNAi reduced DENV/HCV infection and hypothesize that CIDEB can serve as a new host target for treating infections by positive-stranded RNA viruses. We will use CIDEB knockout (KO) models, generated using TAL effector nuclease (TALEN) technology, to determine the importance of CIDEB in the life cycles of various RNA viruses. The results of the proposed studies will not only provide significant insights into the virus- and host-related functions of lipid droplets and CIDEB but also may reveal new therapeutic targets for antiviral intervention directed at medically important RNA viruses.